The immune system has evolved to protect the host from pathogens, while limiting collateral damage to self tissues. This latter process, termed immune tolerance, is enforced by multiple mechanisms. Recessive tolerance is imparted by the deletion or functional inactivation of lymphocytes exhibiting excessive reactivity to self antigens. Alternatively, dominant tolerance is enforced by suppressor cells, such as Foxp3+ regulatory T cells (Tregs), which act in trans to suppress autoreactive lymphocytes. Breakdown of immune tolerance is associated with numerous autoimmune diseases, such as diabetes, lupus, and rheumatoid arthritis. Furthermore, immune tolerance promotes the acceptance of allogeneic transplants, and limits the efficacy of anti-tumor immune therapies. Therefore, there is great interest in defining the mechanisms imparting dominant and recessive immune tolerance, in the hopes that these processes can be manipulated for clinical benefit. Evidence in humans and mice with loss-of-function mutations in autoimmune regulator (Aire) demonstrate a critical role for Aire in the maintenance of immune tolerance. Aire is a transcriptional regulator that promotes the ectopic expression of transcripts preferentially encoding tissue-restricted antigens by epithelial cells in the thymus. Previous studies have demonstrated that the Aire-driven expression of tissue-restricted antigens can induce the deletion of thymocytes reactive to these autoantigens, suggesting that Aire serves a crucial role in enforcing recessive, deletional tolerance. However, our recent work demonstrates that Aire is required for the thymic development of multiple Treg specificities, suggesting that Aire may also function in the establishment of dominant tolerance mediated by a subset of Tregs. Our finding that some, but not all, Tregs are dependent on Aire for development in the thymus suggests that the Treg repertoire may be divided into those that recognize Aire-dependent antigens (Airedep Tregs), and those that recognize Aire-independent antigens (Aireindep Tregs), raising the possibility of a division of labor between the two. The objectives of this proposal are to determine the function of Airedep Tregs in immune tolerance, and to elucidate the role of distinct antigen presenting cell subsets in orchestrating the development and homeostasis of Airedep Tregs. It is our central hypothesis that Airedep Tregs are critical for the maintenance of organ-specific immune tolerance. We will test our central hypothesis by pursuing the following aims. In Aim 1, we will elucidate the functional role of Airedep Tregs in the suppression of autoimmunity, and determine the impact of Aire on the peripheral Treg repertoire. In Aim 2, we will determine the role of antigen presenting cell types in coordinating the thymic development and peripheral homeostasis of Airedep Tregs. The elucidation of these principles will address major gaps in our understanding of the mechanisms that orchestrate the establishment of immune tolerance.